Cytotoxin compounds and methods of isolation

ABSTRACT

The present invention concerns groups of compounds derived from tunicates of the Synoicum species, as well as to pharmaceutical compositions comprising these compounds, and uses thereof. Extracts from tunicates show selective toxicity against several different cancer cell lines in the NCI 60 cell line panel. These compounds are useful in the effective treatment of cancers, particularly malignant melanomas, colon cancer, and renal cancer cell lines.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.14/204,996, filed Mar. 11, 2014, now U.S. Pat. No. 9,394,270, which is acontinuation of U.S. application Ser. No. 12/066,938, filed Aug. 28,2008, now U.S. Pat. No. 8,669,376, which is the National Stage ofInternational Application Number PCT/US2006/036484, filed Sep. 18, 2006,which claims the benefit of U.S. Provisional Application Ser. No.60/717,598, filed Sep. 16, 2005, and US. application Ser. No. 12/066,938is a continuation-in-part of U.S. application Ser. No. 10/906,386, filedFeb. 17, 2005, now U.S. Pat. No. 7,625,885, which claims the benefit ofU.S. Provisional Application Ser. No. 60/521,073, filed Feb. 17, 2004,each of which is hereby incorporated by reference in its entiretyincluding any tables, figures, or drawings.

GOVERNMENT SUPPORT

This invention was made with government support under grant numbersOPP-012518, OPP-9901076, and OPP-0125152 awarded by the National ScienceFoundation. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

One of the greatest efforts of modern medicine is the control andabatement of cellular proliferative disorders, such as cancers.Considerable research has been conducted searching for new biologicallyactive compounds having useful activity for specific cancers and theorganisms which produce these compounds. For example, certain marinesoft corals have shown to be a source of biologically active cytotoxins.Also, compounds from sponges have proven effective againstlipoxygenase-mediated conditions in humans (See U.S. Pat. No.6,750,247).

Tunicates have proven to be an important source of bioactive naturalproducts. Among marine natural products that have advanced as cancertreatments the ecteinascidins and didemnins are derived from tunicates,and the eudistomins have potent antiviral activity. As part of anongoing study of bioactivity among Antarctic marine invertebrates, theinventors had the occasion to study the tunicate Synoicum adareanum.

S. adareanum is a circumpolar tunicate common in the shallow watersaround Anvers island (64° 46′S, 64° 03′W) on the Antarctic Peninsulafrom 15 to 796 meters depth. S. adareanum colonies consist of largerounded or club-shaped heads with the bottom stalk being wrinkled andleathery and only slightly narrower than the head. S. adareanum coloniescan be up to eighteen centimeters high with a diameter of twelvecentimeters. S. adareanum colonies may comprise a single head or, up tosix heads can arise from a single stalk.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns extracts from S. adareanum comprisingPalmerolide A, Palmerolide B, Palmerolide C, Palmerolide D, PalmerolideE, Palmerolide F, Palmerolide G, Palmerolide H, and/or Palmerolide K anduses thereof. Compounds of the invention exhibit bioactivity infield-based feeding-deterrent assays. Presented are novel, isolatedpolyketides, Palmerolide A, Palmerolide B, Palmerolide C, Palmerolide D,Palmerolide E, Palmerolide F, Palmerolide G, Palmerolide H, andPalmerolide K as the major natural product from extracts of S.adareanum. These polyketides display selective cytotoxicity in theNational Cancer Institute (NCI) 60 cell line panel inhibiting, interalia, melanoma (UACC-64, LC₅₀ 0.018 μM) with three orders of magnitudegreater sensitivity relative to other cell lines tested.

The present invention also concerns methods of treating a subject withcancer, comprising administering to the subject a therapeuticallyeffective amount of at least one isolated compound obtained fromextracts of a Synoicum species. In one embodiment, the Synoicum speciesis S. adareanum and the isolated compound is a Palmerolide. In aspecific embodiment, the Palmerolide is chosen from Palmerolide A1,Palmerolide B, Palmerolide C, Palmerolide D, Palmerolide E, PalmerolideF, Palmerolide G, Palmerolide H, and Palmerolide K.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a perspective view of the chemical formula for PalmerolideA.

FIG. 2 shows a chart showing the NMR Data for Palmerolide A.

FIG. 3 shows selected ROE correlations relating the relativestereochemistry between C-11 and C-19.

FIG. 4A shows a chart showing the National Cancer Institute (NCI)Developmental Therapeutics Program In-Vitro Testing Results forPalmerolide A.

FIG. 4B shows a continued chart, showing the National Cancer Institute(NCI) Developmental Therapeutics Program In-Vitro Testing Results forPalmerolide A.

FIG. 5 shows a graph showing National Cancer Institute (NCI)Developmental Therapeutics Program Dose Response Curves for all celllines tested for Palmerolide A.

FIG. 6 shows a graph showing National Cancer Institute (NCI)Developmental Therapeutics Program Dose Response Curves for Melanomacell lines tested for Palmerolide A.

FIG. 7 shows a graph showing National Cancer Institute (NCI)Developmental Therapeutics Program Dose Response Curves for Colon Cancercell lines tested for Palmerolide A.

FIG. 8 shows a graph showing National Cancer Institute (NCI)Developmental Therapeutics Program Dose Response Curves for Renal Cancercell lines tested for Palmerolide A.

FIG. 9 shows a perspective view of the chemical formula for PalmerolideC.

FIG. 10 shows a chart showing the NMR Data for Palmerolide C.

FIG. 11A shows a chart showing the National Cancer Institute (NCI)Developmental Therapeutics Program In-Vitro Testing Results forPalmerolide C.

FIG. 11B shows a continued chart, showing the National Cancer Institute(NCI) Developmental Therapeutics Program In-Vitro Testing Results forPalmerolide C.

FIG. 12 shows a graph showing National Cancer Institute (NCI)Developmental Therapeutics Program Dose Response Curves for all celllines tested for Palmerolide C.

FIG. 13 shows a perspective view of the chemical formula for PalmerolideD.

FIG. 14 shows a chart showing the NMR Data for Palmerolide D.

FIG. 15 shows a perspective view of the chemical formula for PalmerolideE.

FIG. 16 shows a chart showing the NMR Data for Palmerolide E.

FIG. 17A shows a chart showing the National Cancer Institute (NCI)Developmental Therapeutics Program In-Vitro Testing Results forPalmerolide E.

FIG. 17B shows a continued chart, showing the National Cancer Institute(NCI) Developmental Therapeutics Program In-Vitro Testing Results forPalmerolide E.

FIG. 18 shows a graph showing National Cancer Institute (NCI)Developmental Therapeutics Program Dose Response Curves for all celllines tested for Palmerolide E.

FIG. 19 shows the purification steps for isolating Palmerolides A-G.

FIG. 20 shows a chart showing the NMR Data for Palmerolide B.

FIG. 21 shows a chart showing the NMR Data for Palmerolide F.

FIG. 22 shows a chart showing the NMR Data for Palmerolide G.

FIG. 23 shows a chart showing the NMR Data for Palmerolide H.

FIG. 24 shows a chart showing the NMR Data for Palmerolide K.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns extracts from S. adareanum comprisingcompounds, referred to herein as Palmerolides, and uses thereof.Palmerolides specifically exemplified herein include Palmerolide A,Palmerolide B, Palmerolide C, Palmerolide D, Palmerolide E, PalmerolideF, Palmerolide G, Palmerolide H, and Palmerolide K. Compounds of theinvention exhibit bioactivity in field-based feeding-deterrent assays.The subject compounds display selective cytotoxicity in the NationalCancer Institute (NCI) 60 cell line panel inhibiting, inter alia,melanoma cell lines.

Compounds of the present invention include those compounds having thechemical structure shown in formula (I):

wherein:

-   =single or double bond-   R¹ is carboxaldehyde, —CHCHNHC(O)-Alkyl, —OC-Alkyl, —OC-aryl,    —OC-amino, aryl, amino, -vinylamido, arylamido, alkyl, alkoxy,    cycloalkyl, cycloalkoxy, aryloxy, alkylcarbonyl; alkoxycarbonyl,    cycloalkylcarbonyl, cycloalkoxycarbonyl, heteroalkyl,    heterocycloalkyl, heteroaryl, arylcarbonyl, heteroarylcarbonyl,    heterocycloalkylcarbonyl, aryloxycarbonyl, heteroaryloxycarbony,    heterocycloalkoxycarbonyl, a halogen, or —CHO, any of which can be    optionally substituted with H, alkyl, alkoxy, —OH, —NO₂, —NH₂,    —COOH, a halogen, or —CH₃;-   R² is, independently, OH, O-Acyl, carbamate, H, O-alkyl, amino,    —OSO₃H, alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryloxy,    alkylcarbonyl; alkoxycarbonyl, cycloalkylcarbonyl,    cycloalkoxycarbonyl, heteroalkyl, heterocycloalkyl, heteroaryl,    arylcarbonyl, heteroarylcarbonyl, heterocycloalkylcarbonyl,    aryloxycarbonyl, heteroaryloxycarbony, heterocycloalkoxycarbonyl, a    halogen, and/or oxo;-   R³ is H, alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryloxy,    alkylcarbonyl; alkoxycarbonyl, cycloalkylcarbonyl,    cycloalkoxycarbonyl, heteroalkyl, heterocycloalkyl, heteroaryl,    arylcarbonyl, heteroarylcarbonyl, heterocycloalkylcarbonyl,    aryloxycarbonyl, heteroaryloxycarbony, heterocycloalkoxycarbonyl,    halogen, any of which can be optionally substituted with alkyl,    alkoxy, —OH, —NO₂, —NH₂, —COOH, a halogen, and/or —CH₃;-   R⁴ is, independently, H, alkyl, alkoxy, cycloalkyl, cycloalkoxy,    aryloxy, alkylcarbonyl; alkoxycarbonyl, cycloalkylcarbonyl,    cycloalkoxycarbonyl, heteroalkyl, heterocycloalkyl, heteroaryl,    arylcarbonyl, heteroarylcarbonyl, heterocycloalkylcarbonyl,    aryloxycarbonyl, heteroaryloxycarbony, heterocycloalkoxycarbonyl,    halogen, any of which can be optionally substituted with alkyl,    alkoxy, —OH, —NO₂, —NH₂, —COOH, a halogen, and/or —CH₃;-   and isomers, racemates or racemic mixtures thereof, or    pharmaceutically acceptable salts or crystalline forms thereof.

In one embodiment, at least one R² is —OC(NH₂)O.

In one embodiment, R³ is methyl.

In one embodiment, at least one R⁴ is methyl. In a further embodiment,both R⁴ are methyl.

In one embodiment, R¹ is —CHCHNHC(O)CHC(CH₃)₂.

In one embodiment, R¹ is —CHCHNHC(O)CHC(CH₃)CH₂C(CH₃)CH₂.

In one embodiment, R¹ is —CHCH—NHC(O)CH₂C(CH₃)CH₂

In one embodiment, R¹ is —CH═O.

In one embodiment, at least one R² is —OH.

In one embodiment, at least one R² is —OSO₃H.

In one embodiment, at least two R² are —OH and at least one R² is—OC(NH₂)O, and optionally R³ and R⁴ are —CH₃.

Compounds of the present invention include those compounds having thechemical structure shown in formula (II):

wherein:

-   =single or double bond-   R¹ is carboxaldehyde, —CHCHNHC(O)-Alkyl, —OC-Alkyl, —OC-aryl,    —OC-amino, aryl, amino, -vinylamido, arylamido, alkyl, alkoxy,    cycloalkyl, cycloalkoxy, aryloxy, alkylcarbonyl; alkoxycarbonyl,    cycloalkylcarbonyl, cycloalkoxycarbonyl, heteroalkyl,    heterocycloalkyl, heteroaryl, arylcarbonyl, heteroarylcarbonyl,    heterocycloalkylcarbonyl, aryloxycarbonyl, heteroaryloxycarbony,    heterocycloalkoxycarbonyl, a halogen, or —CHO, any of which can be    optionally substituted with H, alkyl, alkoxy, —OH, —NO₂, —NH₂,    —COOH, a halogen, or —CH₃;-   R² is, independently, OH, O-Acyl, carbamate, H, O-alkyl, amino,    —OSO₃H, alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryloxy,    alkylcarbonyl; alkoxycarbonyl, cycloalkylcarbonyl,    cycloalkoxycarbonyl, heteroalkyl, heterocycloalkyl, heteroaryl,    arylcarbonyl, heteroarylcarbonyl, heterocycloalkylcarbonyl,    aryloxycarbonyl, heteroaryloxycarbony, heterocycloalkoxycarbonyl, a    halogen, and/or oxo;-   R³ is H, alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryloxy,    alkylcarbonyl; alkoxycarbonyl, cycloalkylcarbonyl,    cycloalkoxycarbonyl, heteroalkyl, heterocycloalkyl, heteroaryl,    arylcarbonyl, heteroarylcarbonyl, heterocycloalkylcarbonyl,    aryloxycarbonyl, heteroaryloxycarbony, heterocycloalkoxycarbonyl,    halogen, any of which can be optionally substituted with alkyl,    alkoxy, —OH, —NO₂, —NH₂, —COOH, a halogen, and/or —CH₃;-   R⁴ is, independently, H, alkyl, alkoxy, cycloalkyl, cycloalkoxy,    aryloxy, alkylcarbonyl; alkoxycarbonyl, cycloalkylcarbonyl,    cycloalkoxycarbonyl, heteroalkyl, heterocycloalkyl, heteroaryl,    arylcarbonyl, heteroarylcarbonyl, heterocycloalkylcarbonyl,    aryloxycarbonyl, heteroaryloxycarbony, heterocycloalkoxycarbonyl,    halogen, any of which can be optionally substituted with alkyl,    alkoxy, —OH, —NO₂, —NH₂, —COOH, a halogen, and/or —CH₃;-   and isomers, racemates or racemic mixtures thereof, or    pharmaceutically acceptable salts or crystalline forms thereof.

In one embodiment, at least one R² is —OC(NH₂)O.

In one embodiment, R³ is methyl.

In one embodiment, at least one R⁴ is methyl. In a further embodiment,both R⁴ are methyl.

In one embodiment, R¹ is —CHCHNHC(O)CHC(CH₃)₂.

In one embodiment, R¹ is —CHCHNHC(O)CHC(CH₃)CH₂C(CH₃)CH₂.

In one embodiment, R¹ is —CHCH—NHC(O)CH₂C(CH₃)CH₂

In one embodiment, R¹ is —CH═O.

In one embodiment, at least one R² is —OH.

In one embodiment, at least one R² is —OSO₃H.

In one embodiment, at least two R² are —OH and at least one R² is—OC(NH₂)O, and optionally R³ and R⁴ are —CH₃.

In one embodiment, a composition (or an isomer, racemate or racemicmixture thereof, or a pharmaceutically acceptable salt or crystallineform thereof) is provided comprising an isolated Palmerolide A compoundof formula (III):

In yet another embodiment the present invention provides for acomposition (or an isomer, racemate or racemic mixture thereof, or apharmaceutically acceptable salt or crystalline form thereof) comprisingan isolated Palmerolide C compound of formula (IV):

An additional embodiment the present invention provides for acomposition (or an isomer, racemate or racemic mixture thereof, or apharmaceutically acceptable salt or crystalline form thereof) comprisingan isolated Palmerolide D compound of formula (V):

The present invention also provides for a composition (or an isomer,racemate or racemic mixture thereof, or a pharmaceutically acceptablesalt or crystalline form thereof) comprising an isolated Palmerolide Ecompound of formula VI:

The present invention also provides for a composition (or an isomer,racemate or racemic mixture thereof, or a pharmaceutically acceptablesalt or crystalline form thereof) comprising an isolated Palmerolide Bcompound of formula VII:

The present invention also provides for a composition (or an isomer,racemate or racemic mixture thereof, or a pharmaceutically acceptablesalt or crystalline form thereof) comprising an isolated Palmerolide Fcompound of formula (VIII):

The present invention also provides for a composition (or an isomer,racemate or racemic mixture thereof, or a pharmaceutically acceptablesalt or crystalline form thereof) comprising an isolated Palmerolide Gcompound of formula (IX):

The present invention also provides for a composition (or an isomer,racemate or racemic mixture thereof, or a pharmaceutically acceptablesalt or crystalline form thereof) comprising an isolated Palmerolide Hcompound of formula (X):

The present invention also provides for a composition (or an isomer,racemate or racemic mixture thereof, or a pharmaceutically acceptablesalt or crystalline form thereof) comprising an isolated Palmerolide Kcompound of formula (XI):

In preferred embodiments of the invention, the new compounds aresubstantially pure; ideally containing at least 95% of the compound asdetermined by established analytical methods, acceptably containing atleast 90% of the desired compound, permissibly containing at least 75%of the compound.

Also provided by the discoveries of the invention are new pharmaceuticalcompositions between about 0.01% to 60% by weight, preferably 0.1% to50% by weight, and more preferably 1% to 35% by weight based on thetotal weight of the composition, of one of the new compounds of theinvention, or a mixture of two or more such compounds, and one or morepharmaceutically acceptable carriers or diluents.

Those skilled in the art will recognize that the Palmerolide compoundsdisclosed herein can exist in several tautomeric forms. All suchtautomeric forms are considered as part of this invention.

As used herein, alkyl means straight or branched chain, saturated ormono- or polyunsaturated hydrocarbon groups having from 1 to 20 carbonatoms and C_(1-X) alkyl means straight or branched chain alkyl groupscontaining from one up to X carbon atoms, and includes alkyls, alkenyl,and alkynyls. For example, C₁₋₆ alkyl means straight or branched chainalkyl groups containing from 1 up to 6 carbon atoms. Alkoxy means analkyl-O— group in which the alkyl group is as previously described.Cycloalkyl includes a nonaromatic monocyclic or multicyclic ring system,including fused and spiro rings, of from about three to about 10 carbonatoms. A cyclic alkyl may optionally be partially unsaturated.Cycloalkoxy means a cycloalkyl-O— group in which cycloalkyl is asdefined herein. Aryl means an aromatic monocyclic or multicycliccarbocyclic ring system, including fused and spiro rings, containingfrom about six to about 14 carbon atoms. Aryloxy means an aryl-O— groupin which the aryl group is as described herein. Alkylcarbonyl means aRC(O)— group where R is an alkyl group as previously described.Alkoxycarbonyl means an ROC(O)— group where R is an alkyl group aspreviously described. Cycloalkylcarbonyl means an RC(O)— group where Ris a cycloalkyl group as previously described. Cycloalkoxycarbonyl meansan ROC(O)— group where R is a cycloalkyl group as previously described.

Heteroalkyl means a straight or branched-chain having from one to 20carbon atoms and one or more heteroatoms selected from nitrogen, oxygen,or sulphur, wherein the nitrogen and sulphur atoms may optionally beoxidized, i.e., in the form of an N-oxide or an S-oxide.Heterocycloalkyl means a monocyclic or multicyclic ring system (whichmay be saturated or partially unsaturated), including fused and spirorings, of about five to about 10 elements wherein one or more of theelements in the ring system is an element other than carbon and isselected from nitrogen, oxygen, silicon, or sulphur atoms. Heteroarylmeans a five to about a 14-membered aromatic monocyclic or multicyclichydrocarbon ring system, including fused and spiro rings, in which oneor more of the elements in the ring system is an element other thancarbon and is selected from nitrogen, oxygen, silicon, or sulphur andwherein an N atom may be in the form of an N-oxide. Arylcarbonyl meansan aryl-CO— group in which the aryl group is as described herein.Heteroarylcarbonyl means a heteroaryl-CO— group in which the heteroarylgroup is as described herein and heterocycloalkylcarbonyl means aheterocycloalkyl-CO— group in which the heterocycloalkyl group is asdescribed herein. Aryloxycarbonyl means an ROC(O)— group where R is anaryl group as previously described. Heteroaryloxycarbonyl means anROC(O)— group where R is a heteroaryl group as previously described.Heterocycloalkoxy means a heterocycloalkyl-O— group in which theheterocycloalkyl group is as previously described.Heterocycloalkoxycarbonyl means an ROC(O)— group where R is aheterocycloalkyl group as previously described.

Examples of saturated alkyl groups include, but are not limited to,methyl, ethyl, N-propyl, isopropyl, N-butyl, tert-butyl, isobutyl,sec-butyl, N-pentyl, N-hexyl, N-heptyl, and N-octyl. An unsaturatedalkyl group is one having one or more double or triple bonds.Unsaturated alkyl groups include, for example, ethenyl, propenyl,butenyl, hexenyl, vinyl, 2-propynyl, 2-isopentenyl, 2-butadienyl,ethynyl, 1-propynyl, 3-propynyl, and 3-butynyl. Cycloalkyl groupsinclude, for example, cyclopentyl, cyclohexyl, 1-cyclohexenyl,3-cyclohexenyl, and cycloheptyl. Heterocycloalkyl groups include, forexample, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 3-morpholinyl,4-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,2-piperazinyl, and 1,4-diazabicyclooctane. Aryl groups include, forexample, phenyl, indenyl, biphenyl, 1-naphthyl, 2-naphthyl, anthracenyl,and phenanthracenyl. Heteroaryl groups include, for example, 1-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, thiazolyl,pyrazolyl, pyridyl, indolyl, quinolinyl, isoquinolinyl, benzoquinolinyl,carbazolyl, and diazaphenanthrenyl.

As used herein, halogen means the elements fluorine (F), chlorine (Cl),bromine (Br), and iodine (I).

As used herein, oxo refers to compounds containing an oxygen atom, ═O,doubly bonded to carbon or another element. The term embraces aldehydes,carboxylic acids, ketones, sulfonic acids, amides, and esters.

The subject invention also concerns kits, comprising in one or morecontainers a compound or composition of the invention. In a specificembodiment, a kit of the invention comprises one or more of aPalmerolide A, Palmerolide B, Palmerolide C, Palmerolide D, PalmerolideE, Palmerolide F, Palmerolide G, Palmerolide H, and Palmerolide K. Inone embodiment, a kit further comprises a pharmaceutically acceptablecarrier, such as a diluent. In another embodiment, a kit furthercomprises an antitumor or anticancer compound.

The subject invention also concerns methods for inhibiting a vacuolaradenosine triphosphatase (V-ATPase) enzyme, comprising contacting orexposing a V-ATPase to an effective amount of a compound or compositionof the present invention sufficient to inhibit activity or blockfunction of the V-ATPase. In one embodiment, the compound or compositioncomprises one or more Palmerolides selected from Palmerolide A,Palmerolide B, Palmerolide C, Palmerolide D, Palmerolide E, PalmerolideF, Palmerolide G, Palmerolide H, and Palmerolide K.

The subject invention also concerns methods for inhibiting or killing acancer cell, comprising contacting the cell with an effective amount ofa compound or composition of the invention. In one embodiment, thecompound or composition comprises one or more Palmerolides selected fromPalmerolide A, Palmerolide B, Palmerolide C, Palmerolide D, PalmerolideE, Palmerolide F, Palmerolide G, Palmerolide H, and Palmerolide K. Typesof cancer cells that can be inhibited or killed according to the presentinvention include, but are not limited to cancer and/or tumor cells ofbone, breast, kidney, mouth, larynx, esophagus, stomach, testis, cervix,head, neck, colon, ovary, lung, bladder, skin (e.g., melanoma), liver,muscle, pancreas, prostate, blood cells (including lymphocytes), andbrain.

The subject invention also concerns methods for treating a conditionassociated with abnormal expression or overexpression of a V-ATPaseenzyme comprising administering to a person or animal having thecondition and in need of treatment of an effective amount of a compoundor composition of the present invention. Conditions that can be treatedaccording to the present invention include, but are not limited to, cellproliferation disorders, such as cancer; diabetes; pancreatitis; andosteoporosis. In one embodiment, the compound or composition comprisesone or more Palmerolides selected from Palmerolide A, Palmerolide B,Palmerolide C, Palmerolide D, Palmerolide E, Palmerolide F, PalmerolideG, Palmerolide H, and Palmerolide K.

The present invention also provides methods of treating a person oranimal with cancer or an oncological disorder. In one embodiment, amethod comprises administering to the person or animal a therapeuticallyeffective amount of at least one isolated compound obtained fromextracts of a Synoicum species. The animal can be, but is not limitedto, a mammal, such as primate (monkey, chimpanzee, ape, etc.), dog, cat,cow, pig, or horse, or other animals having an oncological disorder.Methods of the invention can optionally include identifying a person oranimal who is or may be in need of treatment of cancer or an oncologicaldisorder. In one embodiment, the Synoicum species is S. adareanum andthe isolated compound is a Palmerolide. In a specific embodiment, thePalmerolide is chosen from Palmerolide A, Palmerolide B, Palmerolide C,Palmerolide D, Palmerolide E, Palmerolide F, Palmerolide G, PalmerolideH, and Palmerolide K. Types of cancer that can be treated according tothe present invention include, but are not limited to cancer and/ortumors of the bone, breast, kidney, mouth, larynx, esophagus, stomach,testis, cervix, head, neck, colon, ovary, lung, bladder, skin (e.g.,melanoma), liver, muscle, pancreas, prostate, blood cells (includinglymphocytes), and brain. For the treatment of oncological disorders, thecompounds and compositions of this invention can be administered to apatient in need of treatment in combination with other antitumor oranticancer substances or with radiation and/or photodynamic therapy orwith surgical treatment to remove a tumor. These other substances orradiation treatments may be given at the same as or at different timesfrom the compounds or compositions of this invention. For example, thecompounds or compositions of the present invention can be used incombination with mitotic inhibitors such as taxol or vinblastine,alkylating agents such as cyclophosamide or ifosfamide, antimetabolitessuch as 5-fluorouracil or hydroxyurea, DNA intercalators such asadriamycin or bleomycin, topoisomerase inhibitors such as etoposide orcamptothecin, antiangiogenic agents such as angiostatin, antiestrogenssuch as tamoxifen, and/or other anti-cancer drugs or antibodies, suchas, for example, GLEEVEC (Novartis Pharmaceuticals Corporation) andHERCEPTIN (Genentech, Inc.), respectively.

The subject invention also concerns methods for isolating and purifyinga compound of the present invention. In one embodiment, the methodcomprises subjecting a Synoicum tunicate to solvent extraction; removingsaid solvent to provide an extract; and fractionating said extract toisolate said Palmerolide.

The subject invention also concerns methods for synthesizing a compoundof the present invention.

“Pharmaceutically acceptable carrier” refers to any carrier, diluent,excipient, wetting agent, buffering agent, suspending agent, lubricatingagent, adjuvant, vehicle, delivery system, emulsifier, disintegrant,absorbent, preservative, surfactant, colorant, flavorant, or sweetener,preferably non-toxic, that would be suitable for use in a pharmaceuticalcomposition.

“Pharmaceutically acceptable equivalent” includes, without limitation,pharmaceutically acceptable salt or crystalline forms, hydrates,metabolites, prodrugs, and isosteres. Many pharmaceutically acceptableequivalents are expected to have the same or similar in vitro or in vivoactivity as the compounds of the invention.

“Pharmaceutically acceptable salt or crystalline form” refers to a saltof the inventive compounds which possesses the desired pharmacologicalactivity and which is neither biologically nor otherwise undesirable.The salt can be formed with acids that include, without limitation,acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate,hexanoate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethane-sulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, thiocyanate, tosylate, andundecanoate. Examples of a base salt include ammonium salts, alkalimetal salts such as sodium and potassium salts, alkaline earth metalsalts such as calcium and magnesium salts, salts with organic bases suchas dicyclohexylamine salts, N-methyl-D-glucamine, and salts with aminoacids such as arginine and lysine. The basic nitrogen-containing groupscan be quarternized with agents including lower alkyl halides such asmethyl, ethyl, propyl, and butyl chlorides, bromides, and iodides;dialkyl sulfates such as dimethyl, diethyl, dibutyl, and diamylsulfates; long chain halides such as decyl, lauryl, myristyl, andstearyl chlorides, bromides, and iodides; and aralkyl halides such asbenzyl and phenethyl bromides.

“Prodrug” refers to a derivative of the inventive compounds thatundergoes biotransformation, such as by metabolism, before exhibiting apharmacological effect. The prodrug is formulated with the objective ofimproved chemical stability, improved patient acceptance and compliance,improved bioavailability, prolonged duration of action, improved organselectivity, improved formulation (for example, increasedhydrosolubility), and/or decreased side effects (for example, toxicity).The prodrug can be readily prepared from the inventive compounds usingmethods known in the art, such as those described by Burger's MedicinalChemistry and Drug Chemistry, Fifth Ed., Vol. 1, pp. 172-178, 949-982(1995).

“Palmerolide,” as used herein, refers to a multi-membered macrocyclicpolyketide bearing carbonate and amide functionality. In one embodiment,the Palmerolide is isolated from the tunicate Synoicum adareanum;collected from the vicinity of Palmer Station on the AntarcticPeninsula.

“Polyketides,” as used herein, refers to any natural compound containingalternating carbonyl and methylene groups (‘β-polyketones’), derivedfrom repeated condensation of acetyl coenzyme A.

“Macrocycle,” as use herein, refers to a large molecule arranged in acircle with various semi-compounds attached at various points. The pointof attachment and the nature of the sub-molecule determine the natureand physiological effect of the compound which contains it.

“Macrolide,” as used herein, refers to a class of antibioticscharacterized by molecules made up of large-ring lactones.

“Olefin,” as used herein, is synonymous with “alkene” and refers to anacyclic hydrocarbon containing one or more double bonds.

As used herein, “a clinical response” is the response of a cellproliferative disorder, such as melanoma, colon, and renal cancer, totreatment with novel compounds disclosed herein. Criteria fordetermining a response to therapy are widely accepted and enablecomparisons of the efficacy alternative treatments (see Slapak and Kufe,Principles of Cancer Therapy, in Harrison's Principles of InternalMedicine, 13^(th) edition, eds. Isselbacher et al., McGraw-Hill, Inc.1994). A complete response (or complete remission) is the disappearanceof all detectable malignant disease. A partial response is anapproximately 50 percent decrease in the product of the greatestperpendicular diameters of one or more lesions. There can be no increasein size of any lesion or the appearance of new lesions. Progressivedisease means at least an approximately 25 percent increase in theproduct of the greatest perpendicular diameter of one lesion or tumor orthe appearance of new lesions or tumors. The response to treatment isevaluated after the subjects had completed therapy.

A “pharmaceutical composition” of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral or nasal (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol, or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates, or phosphates; and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes, or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, orphosphate buffered saline (PBS). In all cases, the composition must besterile and should be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol, and liquid polyetheyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion, and by the use of surfactants. Prevention of the actionof microorganisms can be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol or sorbitol, and/or sodium chloride in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. For administrationby inhalation, the compounds are delivered in the form of an aerosolspray from pressured container or dispenser which contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art. Thecompounds can also be prepared in the form of suppositories (e.g., withconventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art.

Compounds and compositions of the invention can be delivered to a celleither through direct contact with the cell or via a carrier means.Carrier means for delivering compositions to cells are known in the artand include, for example, encapsulating the compound or composition in aliposome moiety. Another means for delivery of a compound of theinvention to a cell comprises attaching the compound to a protein ornucleic acid that is targeted for delivery to the target cell. U.S. Pat.No. 6,960,648 and Published U.S. Patent Application Nos. 20030032594disclose amino acid sequences that can be coupled to another compoundand that allows the compound to be translocated across biologicalmembranes. Published U.S. Patent Application No. 20020035243 alsodescribes compositions for transporting biological moieties across cellmembranes for intracellular delivery. Compounds and compositions canalso be incorporated into polymers, examples of which include poly (D-Llactide-co-glycolide) polymer for intracranial tumors;poly[bis(p-carboxyphenoxy) propane:sebacic acid] in a 20:80 molar ratio(as used in GLIADEL); chondroitin; chitin; and chitosan.

It will be appreciated by those skilled in the art that compounds of theinvention may contain one or more asymmetrically substituted carbonatoms which can give rise to stereoisomers. All such stereoisomers,including enantiomers, and diastereoisomers and mixtures, includingracemic mixtures thereof, are contemplated within the scope of thepresent invention.

A “therapeutically effective amount” is the amount of a Palmerolide ofthe invention, including Palmerolides A, B, C, D, E, F, G, H, and K, orany combination thereof necessary to provide a therapeutically effectiveamount of the corresponding compound in vivo. The amount of the compoundmust be effective to achieve a response, including but not limited tototal prevention of (e.g., protection against and complete cure) and toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms associated with a cellular proliferative diseaseor other indicators as are selected as appropriate measures by thoseskilled in the art. In accordance with the present invention, a suitablesingle dose size is a dose that is capable of preventing or alleviating(reducing or eliminating) a symptom in a patient when administered oneor more times over a suitable time period. One of skill in the art canreadily determine appropriate single dose sizes for systemicadministration based on the size of a mammal and the route ofadministration.

EXAMPLE 1 Hollow Fiber Assay for Preliminary In Vivo Testing

The Biological Testing Branch of the Developmental Therapeutics Programhas adopted a preliminary in vivo screening tool for assessing thepotential anticancer activity of compounds identified by the large scalein vitro cell screen. This hollow fiber based assay has been in usesince June, 1995.

Each compound is tested against a standard panel of 12 human tumor celllines including NCI-H23, NCI-H522, MDA-MB-231, MDA-MB-435, SW-620 COLO205, LOX IMVI, UACC-62, OVCAR-3, OVCAR 5, U251 and SF-295. The celllines are cultivated in RPMI-1640 containing 10% FBS and 2 mM glutamine.On the day preceding hollow fiber preparation the cells are given asupplementation of fresh medium to maintain log phase growth. For fiberpreparation the cells are harvested by standard trypsinization techniqueand resuspended at the desired cell density (varies by cell line between2-10×10⁶ cells/ml). The cell suspension is flushed into 1 mm I.D.polyvinylidene hollow fibers with a molecular weight exclusion of500,000 Da. The hollow fibers are heat-sealed at 2 cm intervals and thesamples generated from these seals are placed into tissue culture mediumand incubated at 37° C. in 5% CO₂ for 24-48 hours prior to implantation.A total of 3 different tumor lines are prepared for each experiment sothat each mouse receives 3 intraperitoneal implants (1 of each tumorline) and 3 subcutaneous implants (1 of each tumor line).

On the day of implantation, samples of each tumor cell line arequantitated for viable cell mass by a stable endpoint MTT assay so thatthe time zero (0) cell mass is known. Thus, the cytostatic and cytocidalcapacities of the test compound can be assessed. Mice are treated withexperimental agents starting on day 3 or 4 following fiber implantationand continuing once daily for a total of 4 doses. Each agent is assessedby intraperitoneal injection at 2 dose levels with 3mice/dose/experiment. Vehicle controls consist of 6 mice receiving thecompound diluent only. The fibers are collected from the mice on the dayfollowing the fourth compound treatment and subjected to the stableendpoint MTT assay. The optical density of each sample is determinedspectrophotometrically at 540 nm and the mean of each treatment group iscalculated. The percent net cell growth in each treatment group iscalculated and compared to the percent net cell growth in the vehicletreated controls. Each compound is assessed in a total of 4 experiments(3 cell lines/experiment×4 experiments=12 cell lines).

Compounds are selected for further testing (for example, time/doseexposure studies preliminary pharmacology studies, subcutaneousxenograft efficacy studies) on the basis of several hollow fiber assaycriteria. These include: (1) a reduction in net cell growth of 50% orgreater in 10 of the 48 possible test combinations (12 cell lines×2sites×2 compound doses); (2) a reduction in net cell growth of 50% orgreater in a minimum of 4 of the 24 distant site combinations(intraperitoneal drug/subcutaneous culture); and/or (3) cell kill of 1or more cell lines in either implant site (reduction in the viable cellmass below the level present at the start of the experiment).

To simplify evaluation, a point system has been adopted which allowsrapid viewing of the activity of a given compound. For this, a value of2 is assigned for each compound dose which results in a 50% or greaterreduction in viable cell mass. The intraperitoneal and subcutaneoussamples are scored separately so that criteria (1) and (2) can beevaluated. Compounds with a combined IP+SC score 20, a SC score 8, or anet cell kill of one or more cell lines can be considered for furtherstudies. The maximum possible score for an agent is 96 (12 cell lines×2sites×2 dose levels×2 [score]). These criteria were statisticallyvalidated by comparing the activity outcomes of >80 randomly selectedcompounds in the hollow fiber assay and in xenograft testing. Thiscomparison indicated that there was a very low probability of missing axenograft active compound if the hollow fiber assay were used as theinitial in viva screening tool. Because of the design of the hollowfiber assay, the results of individual cell lines are not reported sincethe statistical power of the assay is based on the impact of a compoundagainst the entire panel of cells. In addition to the hollow fiberresults, other factors (for example, unique structure, mechanism ofaction, etc.) may result in referral of a compound for further studieswithout the compound meeting these hollow fiber assay criteria.

EXAMPLE II Palmerolide Isolation

Extracts from S. adareanum, Palmerolide A, Palmerolide C, Palmerolide D,and Palmerolide E displayed bioactivity in field-based feeding-deterrentassays, leading the inventors to investigate the chemical nature of theactivity. Presented are novel, isolated polyketides, Palmerolide A,Palmerolide C, Palmerolide D, and Palmerolide E as the major naturalproduct from extracts of S. adareanum. These polyketides displayselective cytotoxicity in the National Cancer Institute (NCI) 60 cellline panel inhibiting, inter alia, melanoma (UACC-64, LC₅₀ 0.018 μM)with three orders of magnitude greater sensitivity relative to othercell lines tested.

In a similar manner, Palmerolides A-H, and K have been isolated to dateusing our standard protocols for fractionation of lipophilic naturalproducts. Freeze-dried biomass was exhaustively extracted withdichloromethane/methanol 1:1 (3×) and the combined extract concentratedto dryness. The extract was applied to a silica gel column packed in100% hexanes. Elution of two column volumes of hexanes as two fractionswas followed by a step gradient of two column volumes of 1:9, 2:8, 4:6,8:2 ethyl acetate/hexanes, 100% ethyl acetate, and finally 5%, 10% and20% MeOH/ethyl acetate, collecting each column volume as a fraction.Repeated high pressure liquid chromatography (HPLC; Waters YMC 5μ, ODS10 mm×30 mm using varying amounts water in MeCN, depending on theisolate) resulted in purification of five palmerolides. (see FIG. 19).Additional palmerolide metabolites can be isolated from the more polarfractions of the lipophilic extract. For example, further fractionationof the ethyl acetate/methanol eluting fractions from flashchromatography of the lipophilic extract has resulted in three morepolar palmerolides (based on inspection of the ¹H NMR spectrum, thesepalmerolides are more highly hydroxylated). Bioassays as describedherein, and other known in the art, can confirm the bioactivity of anyof these isolates.

More polar palmerolides or other melanoma-bioactive compounds present inthe hydrophilic extracts can be fractionated by reversed phase vacuumchromatography. C-18 modified silica gel is packed, in water, in avacuum funnel, then the extract applied and two bed volumes of water,1:9, 2:8, 4:6, 8:2 methanol/water, then 100% methanol pulled throughunder vacuum, collecting one fraction per bed volume. Alternatively,polar constituents can be adsorbed onto polystyrene resins (HP-20 orXAD) and eluted with a similar gradient profile of methanol or acetone.Chromatographic fractions thus obtained are then concentrated andbioassayed. Subsequent or alternative chromatographic steps can beperformed as is well known in the art, utilizing the same stationaryphase described above, over a more narrow solvent gradient, or byanother stationary phase, such as gel permeation (LH-20 or G-10), asnecessary to achieve purification.

Final purification of bioactive isolates is by HPLC. Separations on HPLCutilize any number of stationary phases based on the chromatographicbehavior of the isolate. Typically, reversed-phase (C-18, phenyl)columns work well with non-polar compounds, normal-phase with compoundsof moderate polarity, and the variety of other bonded-phase (amino,cyano, diol) columns work with compounds having character similar to thepacking. However, the use of reversed-phase HPLC on the separations ofeven the most polar organic compounds is not uncommon. We have hadconsiderable success using polymeric stationary phase C-18 (YMC and/orWaters), which accommodate pure water and/or buffer solvents, so thateven the most polar metabolites (amino acids, nucleosides) can beseparated by reversed phase HPLC. Our HPLC instruments are equipped withboth mass-sensitive (differential refractive index (RI) and/orevaporative light scattering (ELSD)) and ultraviolet detectors inseries. Dual detection is important because the RI or ELSD detectors,while relatively insensitive, have a response proportional to quantity.The ultraviolet detector is highly sensitive, but the response isvariable depending on the extinction coefficient of the chromophore.This HPLC instrumentation includes analytical, semi-preparative andpreparative capabilities (four instruments, 0.1 mL/min to 300 mL/min)and our LC/MS can be integrated with any of the instruments to add MS(total ion chromatogram, TIC) detection when necessary, with the addedadvantage of securing mass spectral profiles of the isolates.

Although we have HPLC instrumentation for preparative isolation ofpalmerolides, this methodology has certain drawbacks which can beovercome by using centrifugal countercurrent chromatography (CCC) orcentrifugal partition chromatography (CPC) well known in the art. CCCand CPC have the advantage of being based on the partition of theanalyte between two immiscible phases, a physical property that lendsitself to higher recovery than adsorption chromatography.Instrumentation available today includes multiple volume rotors (bothtechniques utilize a rotary-induced gravity gradient) for developmentalthrough preparative scale separations. Foucault et al. achieved aneffective purification of a polyketide macrolide (amphotericin B) usinga water/DMSO/THF system which works as a good starting point forpalmerolide purification. (Foucault, A. P. et al., 1993).

EXAMPLE III Palmerolide A

Palmerolide A was isolated as a white solid from the 1:1 methanol/ethylacetate fraction eluting from silica gel chromatography of the crudelipophilic (1:1 methanol/dichloromethane) extract. Mass spectroscopicanalysis provided a molecular formula of C₃₃H₄₈N₂O₇ (HRFABMS m/z585.3539, Δ0.1 mmu for [M⁺+1]). The C-1 to C-24 carbon backbone ofpalmerolide A could be unambiguously assigned based on ¹H-¹³Cconnectivity assignments from gHMBC spectra. The macrocycle wascompleted by observation of a correlation between the C-19 (δ 73.69)methine to the C-1 ester carbonyl. Hydroxy methines at C-7 and C-10 wereconclusively assigned based on observation of coupling of the hydroxylprotons in both the gHMBC and COSY spectra: in the gHMBC spectrum, thehydroxy protons correlated to the respective α- and β-carbons, while inthe COSY spectrum correlations were observed between the hydroxylprotons and the hydroxy methine protons. An isopentenoyl amide,established by 2D NMR analysis, completed the gross structure with theexception of CO₂NH₂ remaining unassigned from the molecular formula.This remaining carbon was correlated with the proton on theoxygen-bearing C-11 (δ 75.25), but no further connectivity was evident.The last remaining valence must be occupied by the —NH₂, resulting in acarbamoyl group at C-11 and completing the planar structure ofpalmerolide A.

Stereochemical analysis found all disubstituted olefins to bear the Econfiguration based on their large coupling constants (>14 Hz). Thetrisubstituted olefin at C-16 was also assigned as E based onobservation of a correlation in the ROESY spectrum of H-16 to H₂-18 andof H-15 to H₃-25. Similarly, the C-21 olefin could be assigned the Econfiguration based on ROESY correlations of H-24 to H₃-27, of H-23 toH₃-27 and H-21, and of H₃-27 to H-20 and H₃-26. (R)- and (S)-MTPAesters⁴¹ of palmerolide A demonstrated both C-7 and C-10 to bear the Rconfiguration. Configurational analysis⁴² of the C-10/C-11 fragmentidentified a gauche relationship between H-10 and H-11 based on thesmall ³J_(H-10/H11) observed between the vicinal protons and the large³J_(CH) for both the H-10/C-12 and the H-11/C-9 relationships. Furthersupport for the conformation was found in ²J_(C-11H-10) and²J_(C-10/H-11), both of which were large and negative, defining theabsolute stereochemistry of C-11 as R. Similarly, configurationalanalysis of the C-19/C-20 system suggested an anti relationship of therespective protons based on the large ³J_(H-19/H-20), small³J_(C-21/H-19), ³J_(C-26/H-19) and ³J_(C-18/H-20), as well as the large²J_(C-19/H-20). The relative position of C-18 in this fragment wassecured by the observation of ROESY correlations between H₂-18 and H-20as well as H₂-18 and H₃-26 while no ROESY correlation was observedbetween H₂-18 and H-21, requiring the relative configuration 19R*, 20S*.The NMR data for Palmerolide A can be found in FIG. 2.

The four olefins in the macrocycle constrain the flexibility often foundin macrolides, facilitating stereochemical analysis by NOE studies.Further analysis of the ROESY spectrum revealed the macrolide to adopttwo largely planar sides of a tear-drop shaped cycle, one sideconsisting of C-1 through C-6, the other C-11 through C-19, with C-7through C-10 providing a curvilinear connection. In particular, H-19,H₃-27, H-15 and H₂-13 (see FIG. 3) are sequentially correlated in theROESY spectrum, as are H₃-26, H₂-18, H-16, H-14 and H-12, defining theperiphery of the top and bottom face of the western hemisphere. H-11correlates only to the top series of protons, a result consistent onlywith C-19 and C-11 both adopting the R configuration.

Tunicates are not well known as producers of type I polyketides, thoughthe patellazoles and iejimalides are significant, bioactive,representatives. Palmerolide A is unusual in bearing a small macrocycle,with 20 members, compared to 24 in the patellazoles and iejimalides, anda vinyl amide, a feature more commonly associated withcyanophyte-derived macrolides such as tolytoxin. Palmerolide A displayscytotoxicity toward several other melanoma cell lines, FIG. 2, [M14(LC₅₀0.076 μM), SK-MEL-5 (6.8 μM) and LOX IMVI (9.8 μM)] as well as thepreviously mentioned UACC-62. Besides melanoma, FIG. 3, one colon cancercell line (HCC-2998, 6.5 μM), FIG. 4A, and one renal cancer cell line(RXF 393, 6.5 μM), FIG. 4B, Palmerolide A was largely devoid ofcytotoxicity (LC₅₀>10 μM), representing a selectivity index among testedcell lines of 10³ for the most sensitive cells. Significantly,Palmerolide A is COMPARE.-negative against the NCI database, suggestiveof a previously un-described mechanism of action.

FIGS. 4A and 4B, indicate the National Cancer Institutes DevelopmentalTherapeutics Program In-Vitro Testing Results for Palmerolide A. FIG. 5shows the National Cancer Institute (NCI) Developmental TherapeuticsProgram Dose Response Curves for all cell lines tested for PalmerolideA. In comparison, individual results are shown for Melanoma (FIG. 6),Colon Cancer (FIG. 7) and Renal Cancer (FIG. 8).

EXAMPLE IV Cytotoxiity of Palmerolide C

Palmerolide C, shown below and in FIG. 9, has the chemical formulaC₃₃H₄₉N₂O₇ (for NMR data see FIG. 10). NCI cytotoxicity is shown in FIG.11A and FIG. 11B. NCI Dose Response Curves for all cell lines arepresented in FIG. 12.

EXAMPLE V Cytotoxicity of Palmerolide E

Palmerolide D, shown below and in FIG. 13, has the chemical formulaC₃₆H₅₃N₂O₇. Palmerolide D NMR Data is shown in FIG. 14.

EXAMPLE VI Cytotoxicity of Palmerolide E

Palmerolide E, shown below and in FIG. 15, has the chemical formulaC₂₇H₃₉NO₇ (for NMR data see FIG. 16). NCI cytotoxicity is shown in FIG.17A and FIG. 17B. NCI Dose Response Curves for all cell lines arepresented in FIG. 18.

EXAMPLE VII Bioassay

Established cell lines (UACC-62, SK-MEL-5 and MK14, which are allsensitive to palmerolide A) have been obtained from the NCI Standardprotocols for cell culture are used. UACC-62 (Amundson et al. 2000) andMEL14 (Lin et al., 2003) Cells will be grown in RPMI 1640 mediumsupplemented with 10% fetal bovine serum and glutamine and treated withantibiotics (100 units/mL penicillin, 100 mg/mL streptomycin) in ahumidified atmosphere of 95% air with 5% CO₂ at 37° C.

SK-MEL-5 (Miracco et al., 2003). Cells are grown in Eagle's minimalessential medium with Earle's BSS, adjusted to contain 1.5 g/L sodiumbicarbonate, 0.1 mM non-essential amino acids and 1.0 mM sodium pyruvatein a humidified atmosphere of 95% air with 5% CO₂ at 37° C.

Our bioassays use a 96-well format MTT-based (Vogt et al., 2004) methodfor the quantification of cell growth inhibition or cell lethalitycaused by palmerolides and their derivatives. In this assay,metabolically active cells cleave methylthiazol tetrazolium (MTT), whichis yellow, to form a purple formazan pigment with a concomitantultraviolet shift which can be quantified using a plate readermonitoring absorbance at 540 nm.

EXAMPLE VIII Structure Elucidation of Bioactive Metabolites

Upon isolation of a metabolite, it is important to establish theidentity of the isolate (dereplication). Inspection of the ¹H NMRspectrum, in conjunction with mass spectral information from LC/MSand/or ESIMS, can often lead to identification of previously describedcompounds. Database search and retrieval of marine natural product datagreatly assists in dereplication; databases of marine natural productsproduced by Drs. J. Blunt and M. Munro (Blunt, J. W.; Munro, M. H. G.MarinLit. University of Canterbury, Christchurch, New Zealand, ver 12.4,2004) and mass spectral libraries from NIST and Wiley are available inthis regard. New compounds are subjected to thorough spectroscopicanalysis, as described in the Preliminary Data section for PalmerolideA.

Contemporary spectroscopic methods are employed for structuredetermination of new isolates (Crews et al., 1998; Silverstein et al.,1998). One-dimensional proton and carbon NMR spectroscopy, inconjunction with mass spectral analysis (from LC/MS or electrosprayionization (ESI) or matrix assisted laser desorption ionization (MALDI)mass spectrometers) can secure assignment of the molecular formula of anew compound. Two- and three-bond proton-proton connectivity can beestablished using a combination of two-dimensional NMR techniques suchas COSY (Aue et al., 1976), or extended spin systems with TOCSY(Braunschweiler et al., 1983). Proton-carbon connectivity can beestablished using HMQC or HSQC (Bax et al., 1986a; Bodenhausen et al.,1980) (one bond) and HMBC (Bax et al., 1986b) (two or three bond). TheseNMR experiments are most often acquired in gradient (Maudsley et al.,1978; Ruiz-Cabello et al., 1992) mode for maximal sensitivity, usingsolvent suppression where necessary. If sufficient material isavailable, carbon-carbon connectivity can be established by obtainingthe 2D-INADEQUATE spectrum (Bax et al., 1980); the direct measure of¹³C-¹³C couplings enable significant portions of the structure to bereadily assessed. Stereochemical assignments in conformationallyconstrained systems are based on coupling constant data and the resultsof nuclear Overhauser enhancement or NOESY (Crews et al., 1998;Silverstein et al., 1998) techniques where possible, or byderivatization or degradation. Stereochemical analysis of flexiblesystems (linear or large ring) can be achieved using Murata's method(Murata et al., 1999) of analyzing ³J_(HH) and ^(2,3)J_(CH), derivedfrom decoupling and/or ECOSY (Griesinger et al., 1985) (³J_(HH)) orJ-resolved HMBC (Furihata et al. 1999) (^(2,3)J_(CH)), respectively. Inthe event that spectroscopic methods are not definitive, chemicalderivatization and/or degradation techniques can be employed to furtherclarify the structure or crystals can be grown to facilitate X-raycrystallographic analysis (Yoshida et al., 1995; Ankisetty et al.,2004).

EXAMPLE IX Degradative Studies

The palmerolides provide ample functionalization for degradativestudies. Reductive ozonolysis of palmerolides A, D, and/or E will leadto three hexane polyols (11-13, Scheme 1). Stereochemical conformationof C-7 in these palmerolides is achieved by comparison of the opticalrotation of 11 to that of authentic (R,+)-1,2,6-trihydroxyhexane (Wu etal., 2000).

Positions C-10 and C-11 can be verified by comparison to tetra-ol 12, acompound not reported in the chemical literature. Tetra-ol 12 can bereadily prepared from D-galactose (Scheme 2) based on a scheme modeledafter the preparation of a similar compound (Zhu et al., 2001).Periodate cleavage of the D-galactose 1,3-acetaldehyde acetal (16)(Dolder et al., 1990), followed by homologation via a Wittig reaction,hydrogenation (Zhu et al., 2001) and hydrolysis will provide the desired12 for comparison.

The stereocenters at positions C-19 and C-20 can be confirmed bycomparison of compound 13 to a synthetic sample derived via a previouslypublished route (Scheme 3) (Ohi et al., 1999). Chiral induction in thepublished route was achieved by Sharpless epoxidation of6-methyl-2,6-hepten-1-ol (19), which was treated with methyl lithium tointroduce the methyl group on the opposite face from the alcohol. Thepublished procedure used L-diethyltartrate (L-DET) to achieve the 2S,3Sisomer; the subject preparation requires the 2R,3R isomer, so one beginswith D-diethyltartrate (D-DET). Protected as the acetaldehyde acetal 22,the C-6 alcohol can be oxidized to the ketone which can be homologatedvia Wittig reaction, yielding compound 23, which lacks only deprotectionto make the comparison sample 21.

Degradation studies of palmerolides B (6) and C (7) can similarlyprovide polyols (Scheme 4 and 5). It is expected that C-19 and C-20stereochemistry will be the same in all the palmerolides, so theozonolysis products from palmerolides B and C will provide the sameproduct (13) containing those stereocenters. For palmerolide B, two newdegradation products (24, 25) result. Both enantiomers of 24 are known,from D- and L-glutamic acid (Brunner et al., 1989; Larcheveque et al.,1984), providing a chiroptical comparison once our assignment has beenmade. Product 25, with its terminal triol function, is accessible viaScheme 2 techniques, optionally beginning with an alternate sugar, asnecessary, and homologation with a C₃ Wittig reagent derived from3-bromopropanol.

Penta-ol 26, derived from reductive ozonolysis of palmerolide C (7)(Scheme 5), is prepared from a precursor chosen from the pool of C₅sugars (Scheme 6), as appropriate to address the stereochemistrydetermined spectroscopically.

Schemes 1 through 6 summarize our degradative structure proofs ofpalmerolide A—G stereochemistry. As new palmerolides are isolated theycan be treated accordingly to confirm all stereochemical assignments.

EXAMPLE X Structure-Activity Studies of Palmerolides

The palmerolides exert their melanoma activity by inhibition of vacuolaradenosine triphosphatases (V-ATPases). These are multi-proteintrans-membrane enzymes responsible for translocation of protons out ofthe cytoplasm, resulting in pH regulation of intracellular compartments,a critical function for homeostasis (Sun-Wadaa et al., 2004). They areubiquitous in eukaryotic cells and found occasionally in prokaryotes.The two domains of the enzyme are each composed of roughly half a dozensubunits and ultimately comprise a 20-protein assemblage. The V₀ domain,which is imbedded in the membrane, is the locus of proton transfer whilethe sub-membrane V₁ domain bears catalytic (ATP to ADP) activity (Nishiet al., 2002). In addition to pH regulation, V-ATPases play a role inendocytosis, membrane fusion (Morel et al., 2003), bone resorption(Nomiyama et al., 2005) and other functions which are not simplyfunctions of acidity (Nishi et al., 2002). However, they may havespecialized roles in cancer-regulatory pathways involved in cell growth,differentiation (Martinez-Zaguilan et al., 1993) angiogenesis(Martinez-Zaguilan et al., 1999a), multidrug-resistance (Laurencot etal., 1995; Raghunand et al., 1999; Martinez-Zaguilan et al., 1999b;Sennoune et al., 2004) and metastasis (Martinez-Zaguilan et al., 1998).

Structural studies on V-ATPases which may lead to a better understandingof ligand/receptor interactions include NMR studies of the F subunit(Jones et al., 2001) and more recent X-ray data (Murata et al., 2005) onthe K ring subunit of prokaryotic V-type Na⁺-ATPase, which is homologousto the c/c′/c″ ring of eukaryotic H⁺ V-ATPases. The significance of thisX-ray data is that known inhibitors of H⁺ V-ATPases bind the c subunit(Pàli et al., 2004; Huss et al., 2002), holding the promise of X-raydata on ligand/receptor binding of an inhibitor.

V-ATPases have been implicated in a number of disease states, includingtype 1 diabetes (Myers et al., 2003a; Myers et al., 2003b), osteoporosis(Nomiyama et al., 2005; Sundquist et al., 1990) and several cancers(Sennoune et al., 2004) such as cervical (Ellegaard et al., 1975),breast (Martinez-Zaguilan et al., 1999b) and melanoma (Martinez-Zaguilanet al., 1998). Metastatic breast cancer cells have been demonstrated toshow significant increases in the number of V-ATPase enzymes on theirplasma membranes (Martinez-Zaguilan et al., 1993; Sennoune et al.,2004).Cancer cells require low pH cytoplasm and depend largely on V-ATPases tomaintain that acidity (Martinez-Zaguilan et al., 1993; Raghunand et al.,1999)

Bafilomycin A₁ (4, FIG. 3) is the prototypical V-ATPase inhibitor (Zhanget al., 1994) although its binding and specificity are not nearly asgreat as more recently discovered inhibitors such as salicylihalamides(vis 5) (Erickson et al., 1997; Boyd et al., 2001) oximidines (Kim etal., 1999), lobatamides (McKee et al., 1998; Shen et al., 2002) andpalmerolides. Besides bafilomycins, the concanamycins (Huss et al.,2002), are similarly configured macrocycles while the poecillastrins andchondropsins (Xie et al., 2004) are considerably larger macrolidescomprised of, in addition to a lactone linkage, a lactam linkage.

V-ATPase inhibitors are poised to become a new target of cancerintervention (Chene et al., 2003; Beutler et al., 2003). Key discoveriesleading to first-in-class drugs, such as taxol was for inhibition oftubulin depolymerization (Horwitz et al., 2004), ultimately lead to aproliferation of drugs for the indication and greatly enhance treatmentoptions. Histone deacetylase (HDAC) inhibitors (Arts et al., 2003) aresimilarly undergoing a serge in interest as the Food and DrugAdministration (FDA) has recently approved several Investigational NewDrug (IND) applications. Among several potent V-ATPase inhibitors nowknown it is expected that one will soon emerge as a model to moveforward for IND approval.

The subject functional group manipulation approach begins with the ‘Ruleof 5’ principles elaborated by Lipinski et al., 2001 to addressAbsorption, Distribution, Metabolism, Excretion and Toxicity (ADMET)issues. Among Lipinski's Rules, palmerolides already hold up well: thereare 5 or fewer H-bond donors, MLogP is approximately 1.8 to 2.76(calculated using SimulationsPlus, Inc. (Lancaster, Calif.) ADMETPredictor software) and there are less than 10 H-bond acceptors. Themolecular weights are a bit high, spanning the mid-500's to 610;palmerolide E is the only member less than 500, but it lacks the vinylamide required for V-ATPase activity. Refining our approach with ADMETPredictor, it is clear that solubility and permeability are areas tofocus derivatization studies and the two appear to track in opposingtrends. Thus, solubility improves with the addition of polar functionalgroups while permeability improves with removal of polar functionalgroups. Reconciliation of this dichotomy will take place in thebiological evaluation: compounds prepared according to teachings hereincan be evaluated for melanoma and/or V-ATPase activity, as describedabove, and those retaining sufficient bioactivity (sub-millimolar) anddisplaying promising ADMET properties (based on evaluation by ADMETPredictor) can be subjected to the hollow fiber and/or xenograph assays.

Loss of the C-24 amide results in reduced activity and one alternatearrangement of the C-6 through C-12 hydroxylation/olefination patternalso results in similarly reduced activity, proven by the palmerolides C(7) and E (9), which have undergone NCI 60 cell line bioassay. Otherpalmerolides can be similarly assayed.

For chemical derivatization studies, the palmerolide gross structurewill be divided into three areas of interest: (1) the C-24 terminus andthe associated vinyl amide (SAR1 region, Scheme 7); (2) thehydroxylation/olefination juxtaposition at C-7 through C-12 (SAR2region, Scheme 7); and (3) the group of olefins, which can bemanipulated chemically.

Derivatization studies can be conducted to increase polar groups in eachregion, to decrease polar groups in each region, and to makemodifications without changing the number of polar groups. Permutationsof polar group manipulations on different parts of the core structurelead to other possible modifications. As biological data accumulates,one selects the desired bioactivity profiles (potency and ADMETPredictor properties), rather than merely generating large numbers ofderivatives. Described below are manipulations focused by area as wellas manipulations to add, remove, or leave unchanged, the number of polargroups.

i. The SAR1 Region

The region designated as SAR1 can be evaluated by at least four naturalproducts. Palmerolides A (1), D (8), E (9) and F (10) differ only in thenature of the terminus of the palmerolide parent chain. Syntheticmodifications to be introduced to further probe the C-24 terminus caninclude the following chain terminating groups: Retention of polarfunctional groups:

Non-conjugated:

Conjugated:

Reduction in the number of polar groups:

Addition of polar functional groups:

Non-conjugated:

Conjugated:

Preparation of derivatives 30 to 45 can be accomplished by thecopper-catalyzed vinyl amidation reaction (Scheme 8) (Shen et al.,2000). High yields of stereocontrolled E vinyl amide analogues ofpalmerolides can be prepared by coupling of the desired amide with avinyl iodide 46 using copper (I) thiophenecarboxylate (CuTC) ascatalyst.

The necessary E-vinyl iodide 46 can be prepared in one of two ways. Themost direct route involves chromium-catalyzed (Takai olefination, Takaiet al., 1986) homologation of tri-t-butylsilyl-protected palmerolide E(vis 47, Scheme 9). Note that since functionalization of thepalmerolides is not unlike that of discodermolide (Gunasekera et al.,1990), their stability to conditions described herein can be verified bysynthetic procedures applied to discodermolide as are well known in theart (Paterson et al., 2001; Smith et al., 2000).

Alternately, vinyl iodide 46 can be prepared from Palmerolide A; whilemore steps are involved, this may be desired since Palmerolide A is morepredominant in the tunicate. Thus, palmerolide A can be suitablyprotected (Scheme 10) to differentiate the C-7 and C-10 oxygen functionsfrom the C-11 oxygen function, then the amide selectively hydrolyzed(Eaton et al., 1988) and the resultant phthalimide hydrolyzed to producealdehyde 48. Aldehyde 48 is amenable to direct conversion to the vinyliodide using Danishefsky's method (Di Grandi et al., 1993), the latterof which produces a vinyl iodide directly from a ketone. Thethermodynamically E vinyl iodide (49) results. The Carbamate hydrolyzesunder the conditions employed; the p-methoxybenzyl ether(PMBO(C═NH)CCl₃, PPTS) (Nakajima et al., 1988) protecting group is usedfor that position, to distinguish it in future procedures from the C-7and C-10 oxygenated functions.

If the Danishefsky method (Scheme 9) is undesirable, one can employdegradation of the aldehyde (48) to the protected palmerolide E (47,R=PMB) via Baeyer-Villiger oxidation, hydrolysis of the resultant formylester, then Dess-Martin oxidation (Dess et al., 1991) to the aldehyde(Scheme 11). Intermediate 47 can then be elaborated into the desiredvinyl iodide 46 as shown in Scheme 9. Note that Baeyer-Villiger reagentssuch as those employing Lewis-acid catalysis of trimethylsilylperoxidecan often be accomplished in the presence of olefins without fear ofepoxidation (Brink et al., 2004).

Palmerolide analogues 39-41 can be prepared from the Wittig reaction ofprotected palmerolide E (47) with methyl triphenyl-phosphoniumbromide/BuLi (yielding 39), NaBH₄/CoCl₂ reduction of hydroxymethylderivative 48 (yielding 40) or acetylation of 40 (yielding 41).

ii. SAR2 Region

The C-7 to C-12 SAR2 region can be probed by natural products includingpalmerolides A (1), B (6) and C (7), which differ only in the SAR2region. Palmerolide C is less potent than palmerolide A. Furthersynthetic derivatizations can assess the role of the alcohol groupsbased on modification of template 51 by retention, reduction andaddition of polar functional groups. Alternate modifications are readilyobtained.

SAR2 Modification Template 1 (51)

Retention of polar functional groups:

R₁ R₂ R₃ 52: p-OH Bn H CONH₂ 53: H p-OH Bn CONH₂ 54: p-OH Bn p-OH BnCONH₂ 55: H H H 56: H H p-OH Bn 56A: (CH₃)₂CHCHNHCO H CONH₂ 56B:(CH₃)₂CHCHNHCO (CH₃)₂CHCHNHCO CONH₂

Reduction of polar functional groups:

R₁ R₂ R₃ 57: Me H CONH₂ 58: H Me CONH₂ 59: Me Me CONH₂ 60: H H Me 61: ═OH CONH₂ 62: H ═O CONH₂ 63: CONH₂ H CONH₂ 64: H CONH₂ CONH₂ 65: CONH₂CONH₂ CONH₂

SAR2 Modification Template 2 (66)

Addition of Polar Functional Groups (Template 2):

R₁ R₂ R₃ 67: CONH₂ H CONH₂ 68: H CONH₂ CONH₂ 69: CONH₂ CONH₂ CONH₂

Preparation of derivatives 52-69:

Reaction at C-7 precedes reaction at C-10 (Diyabalanage et al., 2006).Thus C-7 mono-derivatized compounds will be prepared directly. C-10mono-derivatives will be prepared by a protection/deprotection of C-7(tri-t-butylsilyl ether) sequence. The des-carbamato (55) reaction isdescribed in Scheme 10; suitably protected C-7 and C-10 alcohols willprovide access to modification of C-11. Carbamates can be interchangedamong C-7, C-10 and C-11 (Cl₃CC(O)NCHO, then K₂CO₃) (Kocovsky 1986) ifwarranted by bioactivity profiles of the acetates 63-65). Valine esters62 and 63 are modeled after the similar valaciclovir prodrug, whichdemonstrated significantly improved pharmacodynamic properties(Guglielmo et al., 2004). Compounds 67 to 69 can be prepared fromosmimum tetroxide dihydroxylation of 63-65 (forming diastereomericproducts such that two products from each of 63 to 65 will result, afterseparation).

iii. Role of the Olefins

Finally, evaluation of the role of the olefin functions takes advantageof selective hydrogenation catalysts. Perhydrogenation, resulting in 70,is achieved by treating Palmerolide A with H₂, Pd/C, while nickel borideprovides a selective hydrogenation of disubstituted olefins (71) (Choiet al., 1996). Selective reduction of the olefin involved in theα,β-unsaturated carbonyls (vis 72) can be achieved with 10% Pd/C andammonium formate (Ram et al., 1992). Selective reduction of the lonenon-conjugated olefin (73) can be achieved by a number of hydrogenationtechniques, such as with the borohydride exchange resin/Ni₂B catalyst(Yoon 1996). These derivatives of palmerolide A, in addition to otherpalmerolide natural products, which would yield different hydrogenationproducts, provide evidence as to the role of olefins in the bioactivityprofiles of the palmerolides.

iv. Combinations of derivatization

Modifications described for SAR1, SAR2 and/or hydrogenation can readilybe combined if desired by bioactivity profiles and/or ADMET Predictorsimulations.

The total synthesis of natural products is a major tool in thedetermination of the unambiguous structure of a compound. Totalsynthesis also serves as a successful strategy for the formation ofanalogues of the natural product by simple variation of startingmaterials or reactions in the synthetic scheme. Our proposed syntheticstudies toward the palmerolides detail a convergent approach wherebyeach component can be varied to provide analogues inspired by X-rayco-crystallization studies with V-ATPase and by computationalpredictions using ADMET. The synthesis will also be used to confirm thestructure and absolute stereochemistry of the palmerolides. This isespecially important for less abundant palmerolides such as palmerolideD, E, and F due to the trace quantities that can be isolated.

The synthesis is loosely based upon fragments that could be obtainedfrom the degradation studies (shown earlier) using the reductiveozonolysis of palmerolide A. With the degradative studies in mind wedevised a retrosynthetic analysis of palmerolide A (Scheme 12). Ourretrosynthesis divides the molecule into 3 major segments. Themacrocylcic core of palmerolide A should be easily formed by aring-closing metathesis of the precursor 76 (Grubbs et al., 1995). Themetathesis precursor 76 could be formed from the esterification ofalcohol 77 with 78. Fragments 79 and 80 could reasonably be couplingpartners for a Heck reaction to produce the desired alcohol fragment 77(Harris et al., 1996). Amide 80 could be inserted into the C-21 sidechain by means of a copper-catalyzed amidation reaction directly relatedto work the Co-PI helped develop (Shen et al., 2000; Klapars et al.,2001; Jiang et al., 2003). The vinyl iodide 82 required for theamidation reaction could be synthesized by employing the Takaiolefination of the corresponding aldehyde precursor. (Takai et al.,1986) The C-22 double bond could, in turn, be formed by using theSchlosser modification of the Wittig reaction. (Schlosser et al., 1966)This should lead to selective formation of the (E)-alkene. Thestereochemistry of C-20 and C-21 could be established by means of thehighly predictive diastereoselective aldol reaction that was developedby Evans. (Evans et al., 1981) The chiral diol 79 could, in turn, beprepared enantioselectively using the Sharpless asymmetricdihydroxylation (Jacobsen et al., 1988). The asymmetric dihydroxylationcould also be employed to form the chiral alcohol fragment 78. Thisleads to the enantioselective formation of the chiral secondary alcoholon C-8. The vinylogous ester in fragment 78 may be prepared by aHorner-Wadsworth-Emmons olefination reaction (Stocksdale et al., 1998).

The synthesis of palmerolide A begins with the construction of fragment79. Commercially available alkynyl alcohol 85 (Scheme 13) is protectedas the TBDPS ether by treating the alcohol with imidazole and TBDPS-Clin a known procedure (Hanessian et al., 1975). Treatment of alkyne 86with paraformaldehyde and n-BuLi results in the previously knownformation of propargylic alcohol 87 (Nicolaou et al., 1989). Thetreatment of 87 with Red-Al (Sodium bis(2-methoxyethoxy)aluminiumhydride) should result in the formation of the known E-substitutedallylic alcohol 88. Asymmetric dihydroxylation of 88 with Sharplessconditions should lead to the formation of desired diol 89 with highenantioselectivity (Jacobsen et al., 1988). It should be noted at thispoint that the Sharpless asymmetric aminohydroxylation could be employedat this stage of the synthesis (Li et al., 1996). This would lead to theformation of an enantiopure 1,2-amino alcohol. The amide nucleophilegenerally prefers the less hindered carbon of the alkene. Based uponthat observation, the protected amine should be installed on C-11 ofpalmerolide A. This aminohydroxylation route could provide an azavariant of palmerolides. The two secondary alcohol groups of compound 89can be selectively protected as an acetonide upon treatment with acetoneand TsOH (Coe et al., 1989). This would protect the two secondaryalcohols in favor of the primary alcohol due to the thermodynamicstability of the product. At this point, the primary alcohol in 90 canbe protected with a TIPS group (Cunico et al., 1980). The acetonidecould then be opened with FeCl₃ and SiO₂ in chloroform (Kim et al.,1986). This should open the acetonide and not affect the silyl protectedalcohols. The secondary alcohol on C-12 can now be protected with TBS-Cland imidazole. (Corey et al., 1972) The C-12 alcohol protection shouldbe the major product due to the bulk of the TIPS protecting that ispresent on the C-10 hydroxyl group. The C-11 alcohol can now beprotected with MOM-Cl, NaH in THF (Kluge et al., 1972). The ability toprotect all of the hydroxyl groups with different protecting groups isvital to this study because it allows for our main goal, thederivatization of palmerolide A. The aldehyde 92 can be prepared byselectively deprotecting the TBDPS group followed by oxidation withDess-Martin periodinane (Dess et al., 1983). The aldehyde can then beconverted to vinyl iodide 93 via Takai olefination (Jiang et al., 2003).This olefination procedure forms the (E)-vinyl iodide selectively. Thisvinyl iodide would have the correct stereochemistry required for theHeck reaction later in the synthetic route.

The next major step in the synthesis of palmerolide A is the preparationof fragment 78 from the retrosynthesis. The synthesis of 78 begins withthe oxidation of 94 (Scheme 14) with Dess-Martin periodinane (Dess etal., 1983). Sharpless asymmetric dihydroxylation of the alkene wouldlead to the enantioselective formation of diol 95 (Jacobsen et al.,1988). The primary alcohol would be selectively protected with TBDPS-Cland imidazole (Hanessian et al., 1975). The secondary hydroxyl group canbe converted to SEM ether 96 with SEM-Cl and DIPEA in dichloromethane(Lipshutz et al., 1980). The formation of 97 can be accomplished by aHorner-Wadsworth-Emmons olefination reaction (Stocksdale et al., 1998).This would be followed by the deprotection of the TBDPS group with 5 NNaOH in EtOH and oxidation of the alcohol with Dess-Martin periodinane,giving compound 98 (Dess et al., 1983). Petassis olefination of thealdehyde functional group would give olefin 99 (Petassis et al., 1990).Reduction of the ester functional group in compound 99 with DIBAL wouldgive aldehyde 100 (Sunazuka et al., 2000). Oxidation of the aldehydeunder Jones oxidation conditions would lead to the formation ofcarboxylic acid 101 (Bowden et al., 1946). Compound 101 could be useddirectly to form the ester bond later in the synthesis. However,conversion of the acid to the acid chloride with oxalyl chloride 102would lead to a milder esterification.

The last fragment of the molecule that needs to be prepared is the C-17through C-25 amide side chain. The first step (Scheme 15) in thesynthesis of this fragment is the PDC oxidation of the commerciallyavailable geraneol, to yield 103. (Reiter et al., 2003). The use ofEvans diastereoselective Aldol condensation at this point would allowfor the formation of the 104 (Evans et al., 1981). This Evansmethodology has proven to be one of the most dependable tools availableto a synthetic chemist for the formation of syn-selective Aldolproducts. The R group of the oxazolidinone can be varied in order toobtain the product with the highest diastereomeric excess. Protection ofthe alcohol in compound 104 leads to the formation of 105 (Lipshutz etal., 1980). The oxazolidinone auxiliary will then be removed by exposureof 105 to NaOMe in MeOH to lead to the formation of ester 106 (Evans etal., 1981). The reduction of ester 106 with LiAlH₄ followed bysubsequent bromination with PPh₃ and Br₂ should lead to the formation ofbromide 107 (Wiley et al., 1964). The use of the Schlosser variant ofthe Wittig reaction of 107 with 108 should lead to the exclusiveformation of (E)-alkene 109 (Schlosser et al., 1966). Compound 109 couldthen be subjected to the synthetic sequence in Scheme 16 followed bydeprotection of the acetal. This would allow for the synthesis ofpalmerolide E. Deprotection of the acetal followed by Takai olefinationwould lead to compound 110 (Takai et al., 1986; Hagiwara et al., 1987).Copper-catalyzed coupling of the vinyl iodide with the necessary amidewould provide 111 (Shen et al., 2000; Klapars et al., 2001; Jiang etal., 2003). Variation of the amide employed in this coupling could leadto the synthesis palmerolides D and F.

The final stages of the synthesis of palmerolide A would begin (Scheme16) with a Heck coupling of vinyl iodide 93 and alkene 111, which shouldresult in the formation of diene 112 (Harris et al., 1996). Deprotectionof the TIPS group followed by subsequent Petasses olefination wouldprovide compound 113 (Petassis et al., 1990). We are favoring the use ofPetassis olefination due to the methods ability to tolerate the presenceof the functional groups present in compound 112. Deprotection of theSEM group in compound 113 followed by subsequent esterification withcompound 101 should lead to the formation of 114 (Schlessinger et al.,1986). Ring-closing metathesis of 114 using the second-generation Grubbscatalyst could lead to the formation of macrocycle 115 (Grubbs et al.,1995). It has been observed that the metathesis reaction has been usedsuccessfully to close a macrocycle in the synthesis of natural productswith structural similarities to palmerolide A. For example, the RCMapproach in the epothilone family of natural products tends to favor theformation of the (E)-double-bond (Meng et al., 1997). Formation ofcarbamate 116 may be accomplished by treatment of 115 with isocyanicacid and CuCl (Duggan et al., 1989). The final step in the synthesis ofpalmerolide A is the global deprotection of the remaining protectinggroups with MgBr₂ and BuSH in Et₂O (Kim et al., 1991). This is a milddeprotection procedure that should not open the lactone or cleave thecarbamate functional groups present.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated with the scope of the invention without limitation thereto.

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We claim:
 1. A method for isolating a Palmerolide, said methodcomprising: a) subjecting a Synoicum adareanum tunicate biomass tosolvent extraction; b) removing said solvent to provide an extract; andc) fractionating said extract to isolate said Palmerolide.
 2. The methodaccording to claim 1, wherein said solvent is CH₂Cl₂/MeOH.
 3. The methodaccording to claim 1, wherein said extract is fractionated using flashchromatography.
 4. The method according to claim 3, wherein said flashchromatography is gradient flash chromatography over silica.
 5. Themethod according to claim 1, wherein fractions obtained following saidfractionating in step (c) are subjected to gradient elution over silicagel.
 6. The method according to claim 1, wherein fractions obtainedfollowing said fractionating in step (c) are subjected to highperformance liquid chromatography (HPLC).
 7. The method according toclaim 6, wherein said HPLC is reverse phase HPLC.
 8. The methodaccording to claim 1, wherein said solvent is removed by evaporation. 9.The method according to claim 1, wherein after step (b), said extract ispartitioned in a solvent.
 10. The method according to claim 9, whereinsaid solvent is ethyl acetate.
 11. The method according to claim 9,wherein said solvent partitioned extract is washed and dried.
 12. Themethod according to claim 1, wherein said method further comprisesobtaining said tunicate biomass.
 13. The method according to claim 1,wherein the Palmerolide is of the structure of formula I:

wherein:

single or double bond R¹ is carboxaldehyde, —CHCHNHC(O)-Alkyl,—OC-Alkyl, —OC-aryl, —OC-amino, aryl, amino, -vinylamido, arylamido,alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryloxy, alkylcarbonyl;alkoxycarbonyl, cycloalkylcarbonyl, cycloalkoxycarbonyl, heteroalkyl,heterocycloalkyl, heteroaryl, arylcarbonyl, heteroarycarbonyl,heterocycloalkylcarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,heterocycloalkoxycarbonyl, a halogen, or CHO, any of which can beoptionally substituted with H, alkyl, alkoxy, —OH, —NO₂, —NH₂, —COOH, ahalogen, or —CH₃; R² is, independently, OH, O-Acyl, carbamate, H,O-alkyl, amino, —OSO₃H, —OC(NH₂)O—, alkyl, alkoxy, cycloalkyl,cycloalkoxy, aryloxy, alkylcarbonyl; alkoxycarbonyl, cycloalkylcarbonyl,cycloalkoxycarbonyl, heteroalkyl, heterocycloalkyl, heteroaryl,arylcarbonyl, heteroarycarbonyl, heterocycloalkylcarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, heterocycloalkoxycarbonyl, ahalogen, and/or oxo; R³ is H, alkyl, alkoxy, cycloalkyl, cycloalkoxy,aryloxy, alkylcarbonyl; alkoxycarbonyl, cycloalkylcarbonyl,cycloalkoxycarbonyl, heteroalkyl, heterocycloalkyl, heteroaryl,arylcarbonyl, heteroarycarbonyl, heterocycloalkylcarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, heterocycloalkoxycarbonyl,halogen, any of which can be optionally substituted with alkyl, alkoxy,—OH, —NO₂, —NH₂, —COOH, a halogen, and/or —CH₃; R⁴ is, independently, H,alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryloxy, alkylcarbonyl;alkoxycarbonyl, cycloalkylcarbonyl, cycloalkoxycarbonyl, heteroalkyl,heterocycloalkyl, heteroaryl, arylcarbonyl, heteroarycarbonyl,heterocycloalkylcarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,heterocycloalkoxycarbonyl, halogen, any of which can be optionallysubstituted with alkyl, alkoxy, —OH, —NO₂, —NH₂, —COOH, a halogen,and/or —CH₃; and isomers, racemates or racemic mixtures thereof, orpharmaceutically acceptable salts thereof; wherein the Palmerolide doesnot have the structure of


14. The method according to claim 13, wherein said extract isfractionated using reversed phase vacuum chromatography.
 15. The methodaccording to claim 14, comprising applying said extract to a C-18modified silica gel and eluting fractions using water, followed byeluting fractions using a step gradient of methanol/water, and/orfollowed by eluting fractions using methanol under vacuum.
 16. Themethod according to claim 13, wherein said tunicate biomass isfreeze-dried prior to step (a).
 17. The method according to claim 1,wherein the Palmerolide is of the structure of formula II:

wherein:

single or double bond R¹ is carboxaldehyde, —CHCHNHC(O)-Alkyl,—OC-Alkyl, —OC-aryl, —OC-amino, aryl, amino, -vinylamido, arylamido,alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryloxy, alkylcarbonyl;alkoxycarbonyl, cycloalkylcarbonyl, cycloalkoxycarbonyl, heteroalkyl,heterocycloalkyl, heteroaryl, arylcarbonyl, heteroarycarbonyl,heterocycloalkylcarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,heterocycloalkoxycarbonyl, a halogen, or CHO, any of which can beoptionally substituted with H, alkyl, alkoxy, —OH, —NO₂, —NH₂, —COOH, ahalogen, or —CH₃; R² is, independently, OH, O-Acyl, carbamate, H,O-alkyl, amino, —OSO₃H, —OC(NH₂)O—, alkyl, alkoxy, cycloalkyl,cycloalkoxy, aryloxy, alkylcarbonyl; alkoxycarbonyl, cycloalkylcarbonyl,cycloalkoxycarbonyl, heteroalkyl, heterocycloalkyl, heteroaryl,arylcarbonyl, heteroarycarbonyl, heterocycloalkylcarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, heterocycloalkoxycarbonyl, ahalogen, and/or oxo; R³ is H, alkyl, alkoxy, cycloalkyl, cycloalkoxy,aryloxy, alkylcarbonyl; alkoxycarbonyl, cycloalkylcarbonyl,cycloalkoxycarbonyl, heteroalkyl, heterocycloalkyl, heteroaryl,arylcarbonyl, heteroarycarbonyl, heterocycloalkylcarbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, heterocycloalkoxycarbonyl,halogen, any of which can be optionally substituted with alkyl, alkoxy,—OH, —NO₂, —NH₂, —COOH, a halogen, and/or —CH₃; R⁴ is, independently, H,alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryloxy, alkylcarbonyl;alkoxycarbonyl, cycloalkylcarbonyl, cycloalkoxycarbonyl, heteroalkyl,heterocycloalkyl, heteroaryl, arylcarbonyl, heteroarycarbonyl,heterocycloalkylcarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,heterocycloalkoxycarbonyl, halogen, any of which can be optionallysubstituted with alkyl, alkoxy, —OH, —NO₂, —NH₂, —COOH, a halogen,and/or —CH₃; and isomers, racemates or racemic mixtures thereof, orpharmaceutically acceptable salts thereof; wherein the Palmerolide doesnot have the structure of


18. The method according to claim 17, wherein said extract isfractionated using reversed phase vacuum chromatography.
 19. The methodaccording to claim 18, comprising applying said extract to a C-18modified silica gel and eluting fractions using water, followed byeluting fractions using a step gradient of methanol/water, and/orfollowed by eluting fractions using methanol under vacuum.
 20. Themethod according to claim 17, wherein said tunicate biomass isfreeze-dried prior to step (a).
 21. The method according to claim 1,wherein the Palmerolide is Palmerolide B, Palmerolide F, Palmerolide G,Palmerolide H, or Palmerolide K.
 22. The method according to claim 1,wherein following step (b), said extract is subjected to one or morerounds of solvent extraction prior to step (c).
 23. The method accordingto claim 1, wherein said extract is fractionated in step (c) by applyingsaid extract to a silica gel column and eluting fractions using hexane,followed by eluting fractions using a step gradient of ethylacetate/hexane, followed by eluting a fraction using ethyl acetate,and/or followed by eluting fractions using a step gradient of MeOH/ethylacetate.